A variety of attachment chemistries have been developed for the covalent immobilization of probe molecules in DNA or protein arrays (Guo et al., “Direct Fluorescence Analysis of Genetic Polymorphisms by Hybridization with Oligonucleotide Arrays on Glass Supports,” Nucleic Acid Res. 22:5456-5465 (1994); Tomizaki et al., “Protein-Detecting. Microarrays Current,” Chem. Bio. Chem. 6:782-799 (2005); MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination,” Science 289:1760-1763 (2000); Li et al., “Adapting cDNA Microarray Format to Cytokine Detection Protein Arrays,” Langmuir 19:1557-1566 (2003), which are hereby incorporated by reference in their entirety). With few exceptions, for example, thiol-mediated attachment to gold (Bain et al., “Formation of Monolayer Films by the Spontaneous Assembly of Organic Thiols from Solution onto Gold,” J. Am. Chem. Soc. 111:321-335 (1989); Nuzzo et al., “Fundamental Studies of Microscopic Wetting on Organic Surfaces. 1. Formation and Structural Characterization of a Self-Consistent Series of Polyfunctional Organic Monolayers,” J. Am. Chem. Soc. 112:558-569 (1990), which are hereby incorporated by reference in their entirety) and the Staudinger ligation approach (Soellner et al., “Site-Specific Protein Immobilization by Staudinger Ligation,” J. Am. Chem. Soc. 125:11790-11791 (2003), which is hereby incorporated by reference in its entirety), the mechanism of surface attachment is the nucleophilic attack on the surface-bound moiety by the probe molecule of interest (i.e. thiol- or amine-terminated oligonucleotide or protein).
In the case of protein arrays, solution additives are very often required to keep the probe spot hydrated during immobilization (Wang et al., “Microarray-Based Detection of Protein Binding and Functionality by Gold Nanoparticle Probes,” Anal. Chem. 77:5770-5774 (2005)) and to aid in the homogenous distribution of molecules (Deng et al., “Transport at the Air/Water Interface is the Reason for Rings in Protein Microarrays,” J. Am. Chem. Soc. 128:2768-2769 (2006)). This second function of an additive is vital for the removal of “coffee stain” rings (Deegan et al., “Capillary Flow as the Cause of Ring Stains from Dried Liquid props,” Nature 389:827-829 (1997)) and bright center spots, which are presumably the result of the physisorption of molecules from the solution's initial contact with the surface.
Commonly used additives—glycerol, polyethylene glycol, trehalose, and surfactants—unfortunately contain reactive groups themselves (Wu et al., “Comparison of Hydroxylated Print Additives on Antibody Microarray Performance,” J. Proteome Res. 5:2956-2965 (2006)). These include the hydroxyls on glycerol (Olle et al., “Comparison of Antibody Array Substrates and the use of Glycerol to Normalize Spot Morphology,” Exp. Mol. Pathol. 79:206-209 (2005)), trehalose (Kusnezow et al., “Antibody Microarrays: An Evaluation of Production Parameters,” Proteomics 3:254-264 (2003)), polyethylene glycol (Wu et al., “Comparison of Hydroxylated Print Additives on Antibody Microarray Performance,” J. Proteome Res. 5:2956-2965 (2006); Wu et al., “DNA and Protein Microarray Printing on Silicon Nitride Waveguide Surfaces,” Biosensors and Bioelectronics 21:1252-1263 (2006)), and many surfactants (Deng et al., “Transport at the Air/Water Interface is the Reason for Rings in Protein Microarrays,” J. Am. Chem. Soc. 128:2768-2769 (2006); Wu et al., “DNA and Protein Microarray Printing on Silicon Nitride Waveguide Surfaces,” Biosensors and Bioelectronics 21:1252-1263 (2006); Liu et al., “Optimization of Printing Buffer for Protein Microarrays Based on Aldehyde-Modified Glass Slides,” Frontiers in Bioscience 12:3768-3773 (2007)).
In the context of developing methodology for preparing antibody arrays for use with Arrayed Imaging Reflectometry (“AIR”) protein detection technique, it was observed that glycerol in particular interfered with antibody immobilization on glutaraldehyde-coated surfaces. While the precise structure of surface-immobilized glutaraldehyde is not well understood, solution-phase experiments (Migneault et al., “Glutaraldehyde: Behavior in Aqueous Solution, Reaction with Proteins, and Application to Enzyme Crosslinking,” BioTechniques 37:790-802 (2004)) indicate that it is likely polymerized to some extent, providing both saturated and α,β-unsaturated aldehyde functionality for carbonyl- and Michael-addition of reactive amines. Although the reaction of aldehydes with alcohols, such as glycerol, to form hemiacetals and acetals is reversible, the neutral to slightly basic pH employed for protein immobilization provides enhanced stability for acetals (particularly cyclic), while reducing the rate of imine formation (the desired reaction in this case) (Jencks, “Studies on the Mechanism of Oxime and Semicarbazone Formation,” J. Am. Chem. Soc. 81:475-481 (1959)). In fact, as demonstrated in the Examples presented infra, it has been confirmed via NMR spectroscopy that the concentration of glycerol typically employed in protein spotting solutions efficiently hinders reaction between glutaraldehyde and butylamine (a model amine) in MPBS-d at pH 7.2.
Because the amount of immobilized probe correlates with assay performance, and probe spotting becomes inefficient with most currently used additives, to improve assay performance it would be desirable to identify additives that will not participate in a nucleophilic attachment protocol (and circumvent competition for surface reactive groups) but will operate to remove surface morphological anomalies on the resulting chip.
The present invention is directed to overcoming these and other deficiencies in the art.